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Collagen gel-embedding culture of conjunctival epithelial cells

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Abstract

• Background: Collagen has effects on cell morphology, differentiation characteristics and function. Using collagen gel culture, several studies about cell differentiation were reported. In this study, the differentiation of rabbit conjunctival epithelial cells in a collagen gel-embedding culture system was investigated by electron microscope and lectin labeling. • Methods: Rabbit bulbar conjunctival epithelial cells were cultured in type I collagen gel. After I and 2 weeks of culture, some of these cells were stained with PAS and seven kinds of lectins, and others were examined by transmission electron microscopy. • Results: The conjunctival epithelial cells cultured within collagen gel formed stratified cell layers and globules with cavities. The inner layer cells facing the cavities showed PAS and lectin staining patterns similar to those of conjunctival goblet cells in vivo, whereas the staining patterns of the outer layer cells on the collagen matrices resembled the patterns of non-goblet epithelial cells. Microvilli on the surface of the innermost cells, basement membranes beneath the outermost cells, tight junctions, adherent junctions, interdigitating folds and desmosomes between cells were identified on electron microscopic examination. • Conclusion: These results indicate that cell junction structures of the conjunctival epithelial cells are well developed in collagen gel-embedding culture systems, and that the inner layer cells have carbohydrates similar to those of conjunctival goblet cells. Culture of conjunctival epithelial cells within collagen gel is a useful model for examining differentiation of these cells.

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References

  1. Brasitus TA; Goldfarb JP, Hsu KC, Tannenbaum M (1982) Characterization of rat colonic cell surface glycoconjugates by fluoresceinated lectins. I. Importance of fixation techniques. Histochemistry 76:197–209

    Google Scholar 

  2. Chambard M, Gabrion J, Mauchamp J (1981) Influence of collage gel on the orientation of epithelial cell porarity: follicle formation from isolated thyroid cells and from performed monolayers. J Cell Biol 91:157–166

    Google Scholar 

  3. Chen J, Stuckey EC, Berry CL (1985) Three-dimensional culture of rat exocrine pancreatic cells using collagen gels. Br J Exp Pathol 66:551–559

    Google Scholar 

  4. Coulomb B, Dubertret L, Bell E, Merrill C, Fosse M, Breton-Gorius J, Prost C, Touraine R (1983) Endogenous peroxidases in normal human dennis: a marker of fibroblast differentiation. J Invest Dermatol 81:75–78

    Google Scholar 

  5. Freeman JA (1966) Goblet cell fine structure. Anat Rec 154:121–147

    Google Scholar 

  6. Goldstein IJ, Hayes CE (1978) The lectins: carbohydrate binding proteins of plants and animals. Adv Carbohydr Chem Biochem 35:127–340

    Google Scholar 

  7. Haddad A, Smith MD, Herscovics A, Nadler NJ, Leblond CP (1971) Radioautographic study of in vivo and in vitro incorporation of fucose-H3 into thyroglobulin by rat thyroid follicular cells. J Cell Biol 49:856–882

    Google Scholar 

  8. Holly FJ, Lemp MA (1971) Wettability and wetting of corneal epithelium. Exp Eye Res 11:239–250

    Google Scholar 

  9. Holly FJ, Lemp MA (1977) Tear physiology and dry eyes. Surv Ophthalmol 22:69–87

    Google Scholar 

  10. Huang AJW, Tseng SCG, Kenyon KR (1989) Paracellular permeability of corneal and conjunctival epithelia. Invest Ophthalmol Vis Sci 30:684–689

    Google Scholar 

  11. Hynda KK, Klebe RJ, Martin GR (1981) Role of collagenous matrices in the adhesion and growth of cells. J Cell Biol 88:473–485

    Google Scholar 

  12. Kawano K, Uehara F, Sameshima M, Ohba N (1984) Application of lectins for detection of goblet cell carbohydrates of the human conjunctiva. Exp Eye Res 38:439–447

    Google Scholar 

  13. Kawano K, Uehara F, Ohba N (1988) Lectin-cytochemical study on epithelial mucus glycoprotein of conjunctiva and pterygium. Exp Eye Res 47:43–51

    Google Scholar 

  14. Kessing SV (1966) Investigations of the functional mucin. Quantitative studies of the goblet cells of conjunctiva. Acta Ophthalmol (Copenh) 44:439–453

    Google Scholar 

  15. Kinoshita S, Kiorpes TC, Friend J, Thoft RA (1983) Goblet cell density in ocular surface disease. A better indicator than tear mucin. Arch Ophthalmol 101:1284–1287

    Google Scholar 

  16. Kurpakus MA, Stock EL, Jones JCR (1992) The role of the basement membrane in differential expression of keratin proteins in epithelial cells. Dev Biol 150:243–455

    Google Scholar 

  17. McLaughlin B, Caldwell RB, Sasaki Y, Wood To (1985) Freeze fracture quantitative comparison of rabbit corneal epithelial and endothelial membranes. Curr Eye Res 4:951–961

    Google Scholar 

  18. Nandi S, Imagawa W, Tomooka Y, McGrath M, Edery M (1984) Collagen gel culture system and analysis of estrogen effects on mammary carcinogenesis. Arch Toxicol 55:91–96

    Google Scholar 

  19. Nicolaissen B Jr, Eidal K, Hasskjold E, Harsem T, Nass O (1991) Outgrowth of cells from human conjunctival explants onto cornea in vitro. Acta Ophthalmol 69:723–730

    Google Scholar 

  20. Ohji M, Ohmi G, Kiritoshi A, Kinoshita S (1987) Goblet cell density in thermal and chemical injuries. Arch Ophthalmol 105:1686–1688

    Google Scholar 

  21. Opas M, Turksen K, Kalnins VI (1985) Adhesiveness and distribution of vinculin and spectrin in retinal pigmented epithelial cells during growth and differentiation in vitro. Dev Biol 107:269–280

    Google Scholar 

  22. Raviola G (1983) Conjunctival and episcleral blood vessels are permeable to blood-borne horseradish peroxidase. Invest Ophthalmol Vis Sci 24:725–736

    Google Scholar 

  23. Rittig M, Brigel C, Lutjen-Drecoll E (1990) Lectin-binding sites in the anterior segment of the human eye. Graefe's Arch Clin Exp Ophthalmol 228:528–532

    Google Scholar 

  24. Roth J, Berger EG (1982) Immunocytochemical localization of galactosyltransferase in HeLa cells: codistribution with thiamine pyrophosphatase in trans-Golgi cisternae. J Cell Biol 92:223–229

    Google Scholar 

  25. Sarber R, Hull B, Merrill C, Soranno T, Bell E (1981) Regulation of proliferation of fibroblasts of low and high population doubling levels grown in collagen lattices. Mech Ageing Dev 17:107–117

    Google Scholar 

  26. Steuhl P, Rohen JW (1983) Absorption of horse-radish peroxidase by the conjunctival epithelium of monkeys and rabbits. Graefe's Arch Clin Exp Ophthalmol 220:13–18

    Google Scholar 

  27. Tomasek JJ, Hay ED (1984) Analysis of the role of microfilaments and microtubules in acquisition of bipolarity and elongation of fibroblasts in hy drated collagen gel. J Cell Biol 99:536–549

    Google Scholar 

  28. Tomasek JJ, Hay ED, Fujiwara K (1982) Collagen modulates cell shape and cytoskeleton of embryonic cornea and fibroma fibroblasts: distribution of actin, α-actin and myosin. Dev Biol 92:107–122

    Google Scholar 

  29. Tsai RJF, Tseng SCG (1988) Substrate modulation of cultured rabbit conjunctival epithelial cell differentiation and morphology. Invest Ophthalmol Vis Sci 29:1565–1576

    Google Scholar 

  30. Tsai RJF, Ho YS, Chen JK (1994) The effects of fibroblasts on the growth and differentiation of human bulbar conjunctival epithelial cells in an in vitro conjunctival equivalent. Invest Ophthalmol Vis Sci 35:2865–2875

    Google Scholar 

  31. Tsukita S, Oishi K, Akiyama T, Yamanashi Y, Yamamoto T, Tsukita S (1991) Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phos phorylation is elevated. J Cell Biol 113:867–879

    Google Scholar 

  32. Ubels JL, McCartney MD, Lantz WK, Beaird J, Dayalan A, Edelhauser HF (1995) Effects of preservative-free artificial tear solutions on corneal epithelial structure and function. Arch Ophthalmol 113:371–378

    Google Scholar 

  33. Uehara F, Sameshima M, Unoki K, Takumi K, Ohba N (1988) Effects of neuraminidase on lectin binding sites in the corneal and conjunctival epithelium and conjunctival goblet cells of monkey eye. Acta Soc Ophthalmol Jpn 92:1354–1358

    Google Scholar 

  34. Versura P, Maltarello MC, Bonvicini F, Caramazza R, Laschi R (1986) Detection of mucus glycoconjugates in human conjunctiva by using the lectin colloidal gold technique in TEM I. A quantitative study in normal subjects. Acta Ophthalmol 64:445–450

    Google Scholar 

  35. Wang Y, Chen M, Wolosin JM (1993) ZO-1 in corneal epithelium: stratal distribution and synthesis induction by outer cell removal. Exp Eye Res 57:283–292

    Google Scholar 

  36. Watanabe H, Fabricant M, Tisdale AS, Spurr-Michaud SJ, Lindberg K, Gipson IK (1995) Human corneal and conjunctival epithelia in produce a mucin-like glycoprotein for the apical surface. Invest Ophthalmol Vis Sci 36:337–344

    Google Scholar 

  37. Wells PA, DeSiena-Shaw C, Rice B, Foster CS (1988) Detection of ocular mucus in normal human conjunctiva and conjunctiva from patients with cicatrical pemphigoid using lectin probes and histochemical techniques. Exp Eye Res 46:485–497

    Google Scholar 

  38. Wolosin JM, Chen M (1993) Ontogeny of corneal epithelial tight junctions: stratal locale of biosynthetic activities. Invest Ophthalmol Vis Sci 34:2655–2664

    Google Scholar 

  39. Yang J, Nandi S (1983) Growth of cultured cells using collagen as substrate. Int Rev Cytol 81:249–286

    Google Scholar 

  40. Yang J, Richards J, Guzman R, Imagawa W, Nandi S (1980) Sustained growth in primary culture of normal mammary epithelial cells embedded in collagen gels. Proc Natl Acad Sci USA 77:2088–2092

    Google Scholar 

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Authors and Affiliations

  1. Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, 602, Kamigyo-ku, Kyoto, Japan

    Akiko Niiya, Yasuhiro Matsumoto, Takeshi Ishibashi, Keiko Matsumoto & Shigeru Kinoshita

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  1. Akiko Niiya
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  2. Yasuhiro Matsumoto
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  3. Takeshi Ishibashi
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  4. Keiko Matsumoto
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  5. Shigeru Kinoshita
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Niiya, A., Matsumoto, Y., Ishibashi, T. et al. Collagen gel-embedding culture of conjunctival epithelial cells. Graefe's Arch Clin Exp Ophthalmol 235, 32–40 (1997). https://doi.org/10.1007/BF01007835

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  • DOI: https://doi.org/10.1007/BF01007835

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